Composition based upon hydrated lipidic lamellar phases or liposomes containing at least one derivative of labdane

ABSTRACT

The present invention relates to a novel composition based on hydrated lipidic lamelar phases or on liposomes and to a cosmetic or pharmaceutical, particularly dermatological composition incorporating it. 
     According to the invention, the hydrated lipidic lamelar phases or the liposomes contain at least in part a derivative of labdane, or a plant extract containing it. 
     The invention makes it possible to reduce hair drop and to promote regrowth of hair and also makes it possible to prevent gray hair from appearing or to treat gray hair.

The present invention relates to compositions based on hydrated lipidiclamelar phases or on liposomes containing at least one derivative oflabdane, in particular a labd-14-ene, or a plant extract containing it,intended for the preparation of cosmetic or pharmaceutical, particularlydermatological compositions.

Labdanes, and in particular their unsaturated derivatives in 14position, called labd-14-enes, are diterpenes which are fairly widelydistributed in nature.

They are found in particular in different varieties of conifers(particularly pines), tobaccos, cistuses (in particular Cistusladaniferus, Cistus laurifolius), and Coleus (particularly Coleusforskohlii).

Coleus forskohlii represents one of the two hundred or so known speciesof Coleus, belonging to the family of Labiaceae and which are found inthe tropical and subtropical regions of Asia, Africa, Australia and thePacific islands. About nine species are catalogued in India. The tenprincipal varieties of Coleus are catalogued in Indian dictionaries andin particular "The Wealth of India, a dictionary of Indian Raw Materialsand Industrial Products, Raw Materials, volume II, Dehli 1950, pages308-309; the book entitled Indian Materia Medica by Dr. K. M.Nadkarni's, third edition revised and completed by A. K. Nadkarni in twovolumes, Volume I, page 372; the book entitled "The Flora of BritishIndia" by Sir J. D. Hooker, C.B., K.C., S.I. Volume IV entitled"Asclepiadae to Amarantaceae, pages 624 to 627.

The labdane derivatives to which the present invention refers are inparticular derivatives of general formula: ##STR1## in which: R₁ to R₂are identical or different and each represent a hydroxy group or a--O--COR₆ group in which R₆ represents an alkyl, alcoxy or alkenylradical possibly substituted, particularly by one or more hydroxy oramino groups,

R₃ represents an atom of hydrogen or a hydroxy group,

R₄ represents an atom of oxygen or the assembly ##STR2## R₅ representsan ethyl or vinyl radical.

Several derivatives of labdane responding to the above formula have beenextracted from various plants and in particular from the plant Coleusforskolii. Particular reference will be made to documents FR-2 336 138,FR-2 364 211 and EP-A-1-O 243 646 which describe forskoline (compound offormula I in which R₁ =R₃ =OH; R₂ =OCOCH₃ ; R₄ =O; R₅ =CH═CH₂),9-deoxyforskoline (compound of formula I in which R₁ =OH, R₂ =OCOCH₃ ;R₃ =H; R₄ =O; R₅ =CH=CH₂) and coleforsine (compound of formula I Inwhich R₂ =R₃ =OH; R₁ =OCOCH₃ ; R₄ =O; R₅ =CH═CH₂).

Other derivatives of labdane responding to the above formula have beenprepared by synthesis. Particular reference will be made to thefollowing documents: FR-2 372 822; S. V. BHAT et al., J. Chem. Soc.,Perkin Trans. 1982, volume 1, page 767; S V. BHAT et al., J. Med. Chem.1983, volume 26, pages 486-492; EP-A-252 482; EP-A-217 372; E. SEGUIN etal., Planta Medica, 1938, volume 54 No. 1, pages 4-6.

These documents disclose, in addition, that these compounds presentcertain pharmacological properties and in particular a hypotensive andcalming activity on the central nervous system.

Furthermore, document EP-A-O 120 165 describes the local use ofassociations comprising a β-stimulant and an α₂ -inhibitor, for thetreatment of fatty weight. This document cites forskoline by way ofexample of β-stimulant.

The present invention has for its object to solve the new technicalproblem consisting in the preparation of a novel formulation of acosmetic or pharmaceutical, particularly dermatological composition,comprising as active ingredient a labdane or a plant extract containingit and making it possible to potentialize the activity of thisingredient, in particular with respect to the stimulation of regrowth ofhair or the reduction in hair loss.

In fact, it has been discovered in totally unexpected manner, and thisconstitutes the basis of the present invention, that the labdanes or theplant extracts containing them, presented an activity stimulating theregrowth of hair and slowing down hair loss. In addition, it has beendiscovered that this activity, which remains weak for certain of thesecompounds, may be very considerably potentialized by their incorporationin hydrated lipidic lamelar phases or in liposomes.

In this way, according to a first aspect, the present invention concernsa composition based on hydrated lipidic lamelar phases, notablyliposomes, characterized in that said hydrated lipidic lamelar phases,notably liposomes, contain at least in part a derivative of labdane, offormula I hereinafter, and/or a plant extract containing it. ##STR3## inwhich: R₁ and R₂ are identical or different and each represent a hydroxygroup or a --O--CO--R₆ -- group in which R₆ represents an alkyl radicalhaving from 1 to 7 atoms of carbon, alkoxy radical having from 1 to 7atoms of carbon or alkenyl radical having from 1 to 7 atoms of carbon,possibly substituted by one or more hydroxy or ##STR4## groups in whichR₇ and R₈ each represent an atom of hydrogen, an alkyl radical havingfrom 1 to 4 atoms of carbon such as methyl or ethyl or representtogether and with the atom of nitrogen a heterocyclic radical such aspiperidino, morpholino, N'-methyl-piperazino;

R₃ represents an atom of hydrogen or a hydroxy group;

R₄ represents an atom of oxygen or the assembly ##STR5## R₅ representsan ethyl or vinyl radical.

According to a particular characteristic, the labdane derivative is acompound of formula (I) mentioned above in which:

R₁ represents the hydroxy group;

R₂ represents a hydroxy group or a --O--CO--R₆ group in which R₆represents an alkyl radical having from 1 to 4 atoms of carbon, analkoxy radical having from 1 to 4 atoms of carbon or an alkenyl radicalhaving 1 to 4 atoms of carbon, possibly substituted by one or twohydroxy groups or, at one end of chain, by a ##STR6## group in which R₇and R₈ each represent a methyl or ethyl radical or represent togetherand with the atom of nitrogen a heterocyclic radical such as piperidino,morpholino, N'-methylpiperazino;

R₃ represents the hydroxy group;

R₄ represents an atom of oxygen;

R₅ represents the vinyl radical.

The alkyl radicals, represented by R₆, R₇ and R₈ in the above formulaemay be with linear, branched or cyclic chain.

An alkyl radical C₁ -C₇ is for example a methyl, ethyl, propyl,isopropyl, cyclopropyl, isobutyl, butyl, pentyl, neopentyl, preferablymethyl, ethyl or propyl radical.

On the other hand, the alkoxy and alkenyl radicals represented by R₆ inthe above formulae may also be with linear, branched or cyclic chain.

An alkoxy group with C₁ -C₇ is for example a methoxy, ethoxy, propoxy,isopropoxy, cyclopropoxy, butoxy, pentoxy, preferably methoxy, ethoxy orpropoxy group.

An alkenyl group with C₁ -C₇ is for example a vinyl, allyl, isopropenyl,butenyl, pentenyl, cyclohexenyl group.

This labdane derivative is advantageously chosen from the followingcompounds:

n^(o) 1: forskoline

n^(o) 2: 7-O-deacetyl-forskoline

n^(o) 3: coleforsine

n^(o) 4: 7-O-deacetyl-7-β-O-propanoyl-forskoline

n^(o) 5: 7-O-deacetyl-7-β-O-(ethoxycarbonyl)-forskoline

n^(o) 6: 7-O-deacetyl-7-β-O-(propoxycarbonyl)-forskoline

n^(o) 7: 7-O-deacetyl-7-β-O-(2,3-dihydroxy-propanoyl)-forskoline

n^(o) 8: 7-O-deacetyl-7-β-O-(3-methyl-2-butenoyl)-forskoline

n^(o) 9:7,0-deacetyl-7-β-O-[4-(N'-methyl-piperazino)-butanoylΩ-forskoline

n^(o) 10: 7-O-deacetyl-7-β-O-(4-morpholino-butanoyl)-forskoline

n^(o) 11: 6-β-O-[3-piperidino-propanoylΩ-forskoline

n^(o) 12: 6-β-O-(piperidino-acetyl)-forskoline

These labdane derivatives are compounds of formula (I) above in which R₃=OH, R₄ =O, R₅ =CH=CH₃ and R₁ and R₂ have the significances figuring inTable I hereinbelow:

                                      TABLE I                                     __________________________________________________________________________    n.sup.0                                                                           R.sub.1        R.sub.2                                                    __________________________________________________________________________    1   OH             OCOMe                                                      2   OH             OH                                                         3   OCOMe          OH                                                         4   OH             OCOEt                                                      5   OH             OCOOEt                                                     6   OH             OCOOPr                                                     7   OH             OCOCH(OH)CH.sub.2 OH                                       8   OH             OCOCHC(CH.sub.3).sub.2                                     9   OH                                                                                            ##STR7##                                                  10  OH                                                                                            ##STR8##                                                  11                                                                                 ##STR9##      OCOMe                                                      12                                                                                 ##STR10##     OCOMe                                                      __________________________________________________________________________

According to another embodiment of the invention, for preparing such acomposition, an extract containing a labdane derivative as definedpreviously is used, which is for example an extract of Coleus, inparticular an extract of Coleus forskolii, preferably an extract ofroots of the plant Coleus.

Advantageously, it is question of an organic extract of Coleus,particularly of roots of Coleus, preferably obtained by a processcomprising at least one step of extraction with a solvent selected fromthe group constituted by ethyl acetate, methanol, ethanol anddichloromethane.

However, use may be made, as solvent, of organic solvents such asaromatic hydroacarbons, dialkylics, halogenated aliphatic and aromatichydrocarbons, dialkylic ethers, dialkylketones, alkanols, carboxylicacids and their esters; or other solvents such as for exampledimethylformamide, dioxane, tetrahydrofurane and dimethylsulfoxide.

Among the solvents mentioned above, preferred solvents are benzene,toluene or xylene, methylene chloride, chloroform, ethyl acetate,methanol or ethanol.

The ratio of the plant matter with respect to the extraction agent isnot critical and will generally be included between 1:5 and 1:20 partsby weight, and preferably 1:10 parts by weight about.

Extraction is effected at temperatures included between ambienttemperature and the boiling point of the solvent used for extraction.

A particularly advantageous technique of extraction is the so-calledtechnique of extraction employing Soxhlet. It may be advantageous, andin certain cases necessary, to evaporate the solvent, for example bylyophilization, and to take up the crude extracts with a view to apurification.

Within the framework of the present invention, extraction by alcohol isparticularly interesting, particularly at the end of procedure forobtaining the extract due to the usually hardly toxic character of thealcohols. A particularly advantageous alcohol is ethanol.

Another particularly interesting solvent is ethyl acetate because itfurnishes an extract rich in labd-14-ene derivative.

Particular variants of process are also described in the prior art, inparticular in the documents recalled in the preamble of the presentspecification.

The term "lipidic" in the expression "lipidic lamelar phases" covers allthe substances comprising a so-called fatty carbon chain, generallycomprising more than 5 atoms of carbon, this substance usually beingcalled "lipid".

According to the invention, for forming the lipidic lamelar phases,notably liposomes, use is made by way of lipid of amphiphilic lipids,i.e. constituted by molecules presenting a hydrophilic group which isequally well ionic or non-ionic and a lipophilic group, theseamphiphilic lipids being capable of forming lipidic lamelar phases,notably liposomes, in the presence of an aqueous phase, depending on thequantity of water in the mixture.

In particular, among these lipids, mention may be made of: thephospholipids, phosphoaminolipids, glycolipids, polyoxyethylenated fattyalcohols, the possibly polyoxyethylenated polyol esters. Such substancesare for example constituted by an egg or soya lecithin, aphosphatidylserine, a sphyngomyeline, a cerebroside or an oxyethylenatedpolyglycerol stearate.

According to the invention, a lipidic mixture is preferably used,constituted by at least one amphiphilic lipid and at least onehydrophobic lipid such as sterol, like cholesterol or β-sitosterol. Thequantity, expressed in mols, of hydrophobic lipid must generally not begreater than the quantity of amphiphilic lipid, and preferably, it mustnot be greater than 0.5 times this quantity.

The incorporation in hydrated lipidic lamelar phases, notably liposomes,of the compounds or extracts containing these compounds, used inaccordance with the present invention, may be effected in accordancewith known techniques of preparation, described for example in U.S. Pat.No. 4,508,703, and possibly in combination with document U.S. Pat. No.4,621,073.

According to a second aspect, the present invention concerns a cosmeticor pharmaceutical, particularly dermatological composition, intended inparticular to promote regrowth of hair or to reduce hair loss or topromote pigmentation of the epidermis or to prevent gray hair appearingor for treating gray hair, characterized in that it comprises, by way ofactive ingredient, at least one derivative of labdane, of formula Imentioned above, and/or an extract of plants containing it, at leastpartly incorporated in hydrated lipidic lamelar phases, notablyliposomes.

The cosmetic or pharmaceutical, particularly dermatological compositionsaccording to the present invention will generally be produced from thecompositions based on hydrated lipidic lamelar phases, notably liposomesdescribed hereinbefore.

The concentration of labdane derivative or of extract containing it,incorporated at least in part in hydrated lipidic lamelar phases,notably liposomes, will preferably be included between 0.0001% and 1% byweight, preferably still between 0.01% and 0.1% by weight, with respectto the total weight of the cosmetic or pharmaceutical composition.

These proportions are understood to be by dry weight when it is questionof plant extracts.

According to a variant embodiment, a cosmetic or pharmaceutical,particularly dermatological composition according to the inventioncomprises in addition at least one other active substance, at anefficient concentration, selected from xanthines, vitamins, particularlyvitamin B's, tyrosine or its derivatives such as for example glucosetyrosinate, quinine or its derivatives, rubefacients such as methylnicotinate, a supernatant of culture of fibroblasts of papillae, asdefined in document EP-A-272 920, keratin hydrolysates, oligo-elementssuch as zinc, selenium, copper, 5-α-reductase inhibitors such as:progesterone, cyproterone acetate, Minoxidil, azelaic acid and itsderivatives, a 4-methyl-4-azasteroid, in particular17-β-N,N-diethylcarbamoyl-4-methyl-4-aza-5-α-androstan-3-one, or anextract of Serenoa repens, said active substance possibly beingincorporated at least in part in said hydrated lipidic lamelar phases,notably liposomes.

The cosmetic compositions according to the present invention may beapplied topically to promote regrowth of hair, to reduce hair loss, orto promote pigmentation of the epidermis or prevent gray hair fromappearing or for treating gray hair, in particular in compositions inthe form of creams, gels or lotions intended for topical application onthe hair.

Under this aspect, the present invention also provides a process fortreating the hair intended in particular to promote regrowth thereof andto reduce drop thereof or to promote pigmentation of the epidermis orprevent gray hair from appearing or for treating gray hair,characterized in that it comprises the application, in an efficientquantity to produce the effect of regrowth of the hair or for reducingloss thereof, to promote pigmentation of the epidermis or prevent grayhair from appearing or to treat gray hair, of at least one compositionbased on hydrated lipidic lamelar phases, notably liposomes, as definedpreviously, possibly in association with a pharmaceutically orcosmetically acceptable excipient, vehicle or support.

It should be noted that the expression "at least partly incorporated inhydrated lipidic lamelar phases, notably liposomes" is understood tomean in the present description and in the claims, that the derivativeof labdane or the extract of plants containing it is combined withhydrated lipidic lamelar phases, notably liposomes, whatever the form ofthis combination.

However, it is clear that a preferred combination according to theinvention resides in the incorporation, or even the encapsulation, inthe hydrated lipidic lamelar phases, notably liposomes. However, it isnot necessary that the whole of the active principle be incorporated orencapsulated in order to obtain the desired effect.

According to another aspect, the invention further provides a processfor manufacturing a cosmetic or pharmaceutical, particularlydermatological composition intended to promote regrowth of hair or todelay hair loss, to promote pigmentation of the epidermis or to preventgray hair from appearing or to treat gray hair, characterized in that itcomprises in the first place the incorporation of at least one labdaneand/or a plant extract containing it, at least in part in hydratedlipidic lamellar phases, notably liposomes, then the mixture thereof ina pharmaceutically or cosmetically acceptable excipient, vehicle orsupport.

Under this latter aspect, the invention concerns the use of acomposition based on hydrated lipidic lamelar phases, notably liposomessuch as defined hereinabove for the preparation of a cosmetic orpharmaceutical, particularly dermatological composition, intended forthe treatment of hair, particularly for promoting regrowth of the hairor for reducing hair loss and for preventing gray hair from appearing orfor treating gray hair.

Other purposes, characteristics and advantages of the invention willappear more clearly upon reading the following explanatory descriptionmade with reference to several Examples given solely by way ofillustration and which consequently in no way limit the scope of theinvention.

In the Examples, the percentages are expressed by weight, unlessindicated to the contrary.

When it is question of plant extracts, the weights indicated are dryweights unless indicated to the contrary.

EXAMPLE 1

Preparation of a Suspension of Liposomes Containing a Labd-14-eneDerivative

In 25 ml of a mixture of dichloromethane and of methanol in volumetricproportion 4:1, are dissolved 0.398 g of soya lecithin and 0,002 g offorskoline (1α, 6β, 9α-trihydroxy-7β-acetoxy-8,13-epoxy-labd-14-en-11-one).

The solution is evaporated in a rotating flask under reduced pressure atabout 45° C.

The lipidic film obtained is taken up with stirring in about 19.6 ml ofaqueous solution constituted by a phosphate PBS buffer.

A suspension of lipidic vesicles or liposomes is obtained, which is thentreated with ultra-sounds for 15 minutes at 4° C. with a power of 150 W.

A suspension of liposomes is thus obtained, of substantially homogeneoussize, of the order of 139 nm. This suspension contains about 0.01% offorskoline, incorporated in the lipidic phase of the liposomes.

If desired, this suspension may be gelified, for example by mixing itwith an equivalent volume of a gel of Carbopol 940® prepared inconventional manner.

EXAMPLE 2

Obtaining of An Extract of Coleus from Coleus forskolii

60 g of dried, ground roots of Coleus forskolii are extracted withSoxhlet with 600 ml of solvent. Extraction is effected at boilingtemperature of the solvent with an ever-renewed solvent.

The mean yield of extraction is as follows, expressed in dry weight:

a) Ethanol/water mixture (80/20 v/v): about 9.2 to 9.5 g (extract 2A)

b) Ethyl acetate: about 1.5 g (extract 2B)

c) Dichloromethane: about 4 g (extract 2C).

EXAMPLE 3

Incorporation of an Extract of Coleus forskolii in Hydrated LipidicLamelar Phases or in Liposomes

An extract of Coleus forskolii obtained in accordance with Example 2 isincorporated in hydrated lipidic lamelar phases or in liposomes inaccordance with the technique of preparation described in Example 1.

The preparation of the liposomes is for example as follows:

The following are weighed:

    ______________________________________                                        soya lecithin           0.9      g                                            β-sitosterol       0.1      g                                            lyophilized Coleus extract (extract 2B)                                                               0.025    g                                            ______________________________________                                    

These constituents are dissolved in a mixture constituted by 100 ml ofdichloromethane and 25 ml of methanol.

The mixture thus obtained is evaporated in a rotating flask at atemperature of 45° C. under reduced pressure.

The lipidic film thus obtained is then taken up and dispersed withstirring in distilled water qsp 50 g, at ambient temperature for 2hours.

The suspension of lipidic vesicles obtained is then homogenized by anultra-sound treatment for 30 minutes at 4° C., at a power of 150 W.

The mean size of the liposomes thus obtained is about 212 nm.

This suspension is then gelified by mixing it with 50 g of Carbopol 940®gel at 1.25% prepared separately in conventional manner. About 100 g arethus obtained of a gelified suspension of liposomes encapsulating theextract of Coleus forskolii, whose concentration is about 0.025% withrespect to the total weight of the suspension.

EXAMPLES 4 TO 9

By following the experimental process described in Example 1,suspensions of liposomes were prepared from the following compositions:

    ______________________________________                                        Example 4:                                                                    soya lecithin       2           g                                             β-sitosterol   0.05        g                                             Coleus extract (extract 2B)                                                                       0.01        g                                             distilled water, qsp                                                                              50          g                                             Example 5                                                                     lecithin of egg     1.8         g                                             cholesterol         0.1         g                                             extract of Coleus (extract 2A)                                                                    0.02        g                                             distilled water, qsp                                                                              50          g                                             Example 6                                                                     lecithin of soya    2           g                                             extract of Coleus (extract 2C)                                                                    0.035       g                                             distilled water, qsp                                                                              50          g                                             Example 7                                                                     lecithin of soya    2           g                                             β-sitosterol   0.1         g                                             extract of Coleus (extract 2B)                                                                    0.03        g                                             distilled water, qsp                                                                              50          g                                             Example 8                                                                     lecithin of soya    2           g                                             progesterone        0.001       g                                             extract of Coleus (extract 2A)                                                                    0.0275      g                                             distilled water, qsp                                                                              45          g                                             Example 9                                                                     lecithin of soya    2           g                                             extract of Coleus (extract 2C)                                                                    0.025       g                                             extract of Serenoa repens                                                                         0.01        g                                             distilled water     50          g                                             ______________________________________                                    

EXAMPLE 10

Evidencing of an Hair Loss Preventing Activity on the Growth of the Hair

The hair loss preventing activity on the growth of the hair is evidencedby studying the activity of the products according to the invention onthe pilary cycle of Sprague Dawley rats aged 23 days. The rats areshaved, in the lower part of their back, on the 24th day.

From the 25th day and up to the 65th day, 6 days out of 7, the productsto be tested are then applied at a dose ranging from 0.5 ml to 2 ml as afunction of the weight gain of the animals.

At substantially regular intervals of time (every 3 days about), a smalltuft of at least 10 hairs is removed, with tweezers, on the left-handside of the animal: at the level of the flank.

The percentage of hairs in anagenous phase (phase of growth) is thencounted as a function of time. Identification of the hairs in anagenousphase is effected by microscopic observation of the lower end of thehair. The base of the hairs in anagenous phase is fine, as the livingpart has remained in the dermis, whilst the base of the hairs intelogenous phase (rest phase) is in club form. The hairs in catagenousphase (intermediate phase of regression) are always very few.

The study is made on 30 rats distributed in 3 batches of 10 animals. Thefirst batch receives the product of Example 3. The second receives analcoholic solution of the same extract of Coleus at the sameconcentration of 0.025%. The third batch is the control batch receivingno product.

The results of this trichokinetic study are given in the accompanyingsingle FIGURE.

In this FIGURE, the percentage of hairs in anagenous phase is plotted onthe y-axis and the number of days on the x-axis.

The curve joining the triangles whose apex is directed upwardlycorresponds to the results obtained with liposomes incorporating 0.025%of extract of Coleus, and prepared according to Example 3.

The curve joining the triangles whose apex is directed downwardlycorresponds to the results obtained with the control batch.

The curve joining the circles corresponds to the results obtained withan extract of Coleus in alcohol, in the free state, at the sameproportion of 0.025% by weight, with respect to the total weight of thecomposition.

It will be observed that the number of hairs in anagenous phaseincreases much more rapidly in the batch receiving the extract of Coleusin liposomes according to the invention, than in the one receiving thealcoholic solution of extract of Coleus (respectively 65% and 25% on day37). It will also be observed that the anagenous phase extends longer.

On the other hand, if the curve corresponding to the alcoholic solutionof extract of Coleus is compared with the control curve, no reallysignificant difference is observed.

In this way, it is clear that the extracts of Coleus incorporated in theliposomes, according to the invention, by the extension of the durationof the anagenous phase, very clearly slow down hair loss and promoteregrowth thereof.

EXAMPLE 11

Measurement of the Pigmenting Activity of an Extract of Coleusforskohlii Incorporated in Liposomes on the Cutaneous Pigmentation inthe Guinea Pig

Protocol

The study was made on a batch of 10 three-coloured guinea pigs.

Before and during the experimentation, the right and left flanks of theguinea pigs were carefully shaved, every day for the first 5 days(period of exposure to U.V.), then every 2 days until the end of thestudy.

For each animal, there are determined on each flank comparably pigmentedmarks, most often of light brown appearance. On one of the two flankstaken at random, about 0.5 g of product to be tested or of controlproduct, depending on the batch, is applied 10 mins. before exposure toultraviolet rays, the other flank being exposed "bare" by way ofcontrol.

The application of the products to be tested is effected as from thefirst day of exposure until the animal is sacrificed.

Exposure to ultraviolet radiation is effected by means of a solarsimulator delivering 86% of U.V.A. and 14% U.V.B during the first 5 daysof the experiment, at a rate of 5 mins. the first day, 10 mins. thesecond day, 15 mins. the third day and 20 mins. the fourth and fifthdays.

The animals are sacrificed 12 days after the last exposure, and acutaneous biopsy is effected.

A fragment of skin is thus removed from the non-treated but exposedflank as well as from the other treated and exposed flank.

A histological examination is then made of the cutaneous fragments.

This examination comprises: on the one hand, the study of themelanogenesis by the Argentaffine de Fontana method on sections of 4>m(Techniques d'histologie, Professor Chevreau, Ed. Maloine, 1977, page157), on the other hand, the assessment of the thickness of theepidermis on sections of 4>m coloured in accordance with the trichomicmethod of Masson.

The study of melanogenesis by the Argentaffine de Fontana reactionsinvolves the precipitation of black metallic silver from solutions ofsilver salts under the influence of reducing substances, which, for thepurposes of this application is directed toward pigments as a particularembodiment. The process can be conducted in various ways using differentsilver salts at an alkaline, neutral or acid pH. The most commonly usedprocess utilizes ammoniacal silver nitrate in the process. The sectionunder consideration is generally already waxed and then is dewaxed andrehydrated at the time of use. The section is treated for a ten minuteduration with an iodine solution containing one gram of iodine and twograms of potassium iodide in 300 ml of water. The section is then fullywashed with distilled water for a period ranging from about 15 minutesto about 2 hours. The section is then immersed into an ammoniacal silvernitrate solution of Fontana in a closed cylinder in the absence of lightfor a period of 18 to 24 hours.

The solution of Fontana noted above is prepared by the dropwise additionof concentrated ammonia to 20 ml of 10% silver nitrate under constantstirring up to the moment the precipitate, which is at first formed, ispractically fully dissolved again.

Subsequently, 20 ml of distilled water is added to the solution and itis allowed to stand for 24 hours. The solution is then decanted into adark flask and made available for use. The solution of Fontana preparedas noted above can be stored for about one month. The solution isfiltered before each use.

The "Lison" variation can be used instead of Fontana. To prepare theLison variant, ammonia solution is added dropwise to a solution of 5%silver nitrate up to the point when the precipatate formed isredissolved. Then very carefully, add dropwise a 5% silver nitratesolution up to the appearance of persistent turbidity. The liquid shouldno longer emit an ammonia odor. Decant before use.

The treated section is then rinsed with distilled water. Subsequently,change the color in the solution adding 0.1% gold chloride during aperiod of about 4 minutes. Then rinse with distilled water and fix in asolution of 5% sodium hyposulfite over a two minute period. Add water,then perform a nuclear coloration with Kernechtroi during a 5 minuteperiod. Pass with alcohol, xylenes and embed with balm.

The result of the method is that the reducing substances are colored inblack. The method is operative on such pigments as melanines,lipofuscines, pigments of the illness of Dubin-Johnson and bilirubin.

The Trichromic Coloration of Masson also referred to above consists ofcombining a nuclear coloration by hemalun (or preferably trioxyhemateinor slow hematoxylin), a cytoplasmic coloration by an admixture of aciddyes (acid fuchsin-xylidin scarlet) and an elective coloration ofcollagen by another acid dye (aniline blue or light green).

Once again the dewaxed section is rehydrated and the nuclei is coloredin one of the following dyes: either in hemalun for a period of tenminutes or more, in ferric trioxyhemateine on slides during 1-3 minutes,or (in the event of fine or delicate cytological research) with slowferric hematoxylin for about 24 hours.

The section is washed with water and in the case of coloration withferric trioxyhemateine with tap water for approximately 2-5 minutes. Thecytoplasms are colored with a mixture of acid fuchsin-xylidin scarlet(which mixture consists of the one part aqueous solution of 1% of acidfuchsin and two parts of a solution of 1% xylidin-scarlet in acidicwater at 1%) for a period of 5 minutes. The section is rinsed withdistilled water and immersed in an aqueous solution of 1%phosphomolybdic acid for about 5 minutes. The slide containing thesection is then removed and without washing, indicators of aniline blueor light green are contacted with the sample and allowed to react for aperiod of 1-5 minutes, the times being variable depending upon the dyeand the particular material to be studied.

To prepare aniline blue dye, 100 ml of distilled water are brought to aboil and 2-3 grams of aniline blue is added thereto. The solution iswithdrawn from the heat source and 2.5 ml of acetic acid are added. Thecontainer holding the system is closed with cotton, the solution cooledand filtered.

The light green dye solution is made by mixing 1 gram of light green in100 ml of distilled water and 1 ml of glacial acetic acid.

The section sample is then contacted with acetic acid at 1% for about 5minutes or more depending upon the dye used. Immerse the sample directlyinto to successive baths of (absolute alcohol, Xylenes I, II.) Thesample is then embedded with balm.

The results of the coloration are as follows:

nuclei: blue-black or brown, according to the nuclear dye used;

cytoplasms: pink to red; red blood corpuscles: sharp red;

collagen: blue or green (according to the acid dye used);

The aniline blue often gives a coloration slightly deeper or morethickened, but it allows one to better distinguish the finest fibrillaeof collagen. The green is more transparent and fades more easily underlight. Additionally, mucous: blue or green; keratin: sharp red; andresilient slides: pink.

The study of the thickness of the epidermis and of the intensity of themelanogenesis makes it possible to assess a tanning effect or moreexactly the activation of the process of melanogenesis.

To study the melanogenesis, two zones taken at random from a pigmentedmark are examined, in which 25 malpigian cells are noted and among thelatter the "activated" melanocytes are counted, i.e. containing melaninein a cluster. The activation of the process of melanogenesis is thenexpressed in percentage of activated cells from the average of these twovalues.

On these same zones, the quantity of melanine in the other layers of theepidermis is examined and this quantity is assessed overall in a scalewith five values varying from 0 to 4 depending on whether the quantityof melanine formed is zero, low, average, large or very large.

Table I shows the results of the histological study giving thepercentage of activation and making it possible to assess the variationsin the quantity of melanine formed (average values on the scale definedhereinabove), of the thickness of the epidermis (expressed in >m).

The product tested is constituted by an extract of Coleus with ethylacetate according to Example 2b, incoporated in liposomes according toExample 3, but titrating 0.0284% of Coleus extract instead of 0.025%.

                  TABLE I                                                         ______________________________________                                                           Control                                                                              Treated                                                                flank  flank                                               ______________________________________                                        % Activation         33.40    85.30                                           Quantity of melanine 0.16      2.39                                           Thickening of the epidermis in >m                                                                  8.32     14.10                                           ______________________________________                                    

From Table I it may be ascertained that the extract of Coleusincorporated in liposomes according to the invention is active onmelanogenesis. A reading of the histological sections of the treatedflanks shows a very high rate of activated melanocytes presentingrhizomic forms, and a considerable migration of the grains of melaninesin the epidermis.

These results therefore clearly confirm the activity of the extracts ofColeus incorporated in liposomes according to the invention on theactivation of the melanocytes.

Various examples of formulation of cosmetic or pharmaceuticalcompositions promoting regrowth of the hair and/or reducing hair dropand/or preventing gray hair from appearing or for treating gray hair,will be given hereinafter. The hydrosoluble constituents mayadvantageously be dissolved in the aqueous phase in which the lipidicpowder is dispersed, as indicated in Example 3. For example, theseconstituents may be incorporated, at least in part, in the liposomes.

EXAMPLE 12

Lotion Reducing Hair Loss

    ______________________________________                                        Suspension of liposomes according to Example 4                                                         50       g                                           Carbopol 940 ®       0.03     g                                           distilled water, qsp     100      ml                                          ______________________________________                                    

This lotion is applied on the scalp twice a day for 6 months.

EXAMPLE 13

Anti-hair Drop Lotion

    ______________________________________                                        Suspension of liposomes according to Example 5                                                         50       g                                           Carbopol 940 ®       0.03     g                                           panthenol                0.1      g                                           keratin hydrolysat       0.2      g                                           hydrosoluble perfume     0.1      g                                           distilled water, qsp     100      ml                                          ______________________________________                                    

This lotion is applied on the scalp twice a day for 6 months.

EXAMPLE 14

Lotion for Stimulating the Dermal Papilla

    ______________________________________                                        Suspension of liposomes according to Example 6                                                         50       g                                           Carbopol 940 ®       0.04     g                                           Phytantriol ®        0.1      g                                           conserving agents        0.05     g                                           protein-zinc complex     0.1      g                                           water, qsp               100      ml                                          ______________________________________                                    

This lotion is applied on the scalp, over the areas where the hair hasfallen, 5 days out of 7, in a cure of 6 months. This application isfollowed by a slight massage.

EXAMPLE 15

Lotion Promoting Regrowth of the Hair and Preventing Gray Hair fromAppearing

    ______________________________________                                        Suspension of liposomes according to Example 7                                                         50       g                                           Carbopol 940 ®       0.05                                                 glucose tyrosinate       0.05                                                 complex of oligo-elements                                                                              0.1                                                  theophylline             0.01                                                 conserving agents        0.05                                                 distilled water, qsp     100      ml                                          ______________________________________                                    

This lotion is applied in the evening on the scalp over the grayingareas and the zones where the hair has fallen, in a cure of 4 months.

EXAMPLE 16

Hair Loss Preventing Gel

    ______________________________________                                        Suspension of liposomes according to Example 8                                                         45       g                                           perfume                  0.1      g                                           Carbopol 940 ® gel at 1.5%                                                                         50       g                                           distilled water, qsp     100      g                                           ______________________________________                                    

Apply this gel, preferably after a shampoo, twice a week, in a cure of 2months.

EXAMPLE 17

Hair Loss Preventing Gel Against Seborrheic Alopecia

    ______________________________________                                        Suspension of liposomes according to Example 9                                                         50       g                                           perfume                  0.1                                                  protein-zinc complex     0.05                                                 Carbopol gel at 1.5%     45       g                                           water, qsp               100      g                                           ______________________________________                                    

This gel is preferably applied every evening over the areas of drop, ina cure of 4 months.

EXAMPLE 18

Lotion Reducing Hair Loss Preventing

    ______________________________________                                        Suspension of liposomes according to Example 1                                                         20       g                                           Carbopol 940 ®       0.03     g                                           distilled water, qsp     100      ml                                          ______________________________________                                    

EXAMPLE 19

Tanning Gel

    ______________________________________                                        Suspension of liposomes (non-gelified) according to                                                     50      g                                           Example 3                                                                     Carbopol 940 ®        1.25    g                                           hydrosoluble solar filter 6       g                                           distilled water, qsp      100     g                                           ______________________________________                                    

EXAMPLE 20

Lotion Promoting Regrowth of the Hair

    ______________________________________                                        Suspension of liposomes according to Example 7 but                                                        50     g                                          containing in the aqueous phase human keratin                                 hydrolysate at a concentration of 5%                                          Carbopol 940.sup.R          0.05   g                                          Complex of oligo-elements   0.1    g                                          conserving agent            0.05   g                                          distilled water, qsp        100    ml                                         ______________________________________                                    

The suspension of liposomes according to Example 7 containing in theaqueous phase human keratin hydrolysate at 5%, is prepared in accordancewith Example 3 except that the lipidic film is taken up in an aqueoussolution containing 5% of human keratin hydrolysate.

This solution is applied in the evenings on the scalp in the areas ofhair loss, in a cure of 4 months.

As it is well known to those skilled in the art, liposomes represent aparticular embodiment of hydrated lipidic lamelar phases. Thus,according to the invention, the hydrated lipidic lamelar phases are in aspecific embodiment in the form of liposomes.

We claim:
 1. A method for promoting pigmentation of the epidermiscomprising applying on the desired area, an effective amount to promotepigmentation of the epidermis of at least one composition comprisinghydrated lipidic lamellar phases or liposomes containing at least inpart, a component selected from a derivative of labdane, of formula Ihereinbelow: ##STR11## in which: R₁ and R₂ are identical or differentand each represent a hydroxy group of an --O--CO--R₆ group in which R₆represents an alkyl radical having from 1 to 7 atoms of carbon, alkoxyradical having from 1 to 7 atoms of carbon or alkenyl radical havingfrom 1 to 7 atoms of carbon, optionally substituted by one or morehydroxy or ##STR12## groups in which R₇ and R₈ each represent an atom ofhydrogen, an alkyl radical having from 1 to 4 atoms of carbon or R₇ andR₈ represent together and with the atom of nitrogen, a heterocyclicradical such as piperidino, morpholino, N'-methyl-piperazino;R₃represents an atom of hydrogen or a hydroxy group; R₄ represents an atomof oxygen or the assembly ##STR13## R₅ represents an ethyl or vinylradical; optionally in a pharmaceutically or cosmetically acceptableexcipient.
 2. The method defined in claim 1 wherein the labdanederivative is a compound of formula I in which:R₁ represents the hydroxygroup; R₂ represents a hydroxy group or a --O--OH--R₆ group in which R₆represents an alkyl radical having from 1 to 4 atoms of carbon, analkoxy radical having from 1 to 4 atoms of carbon or an alkenyl radicalhaving from 1 to 4 atoms of carbon, possibly substituted by one or twohydroxy groups or, at one chain end, by a ##STR14## group in which R₇and R₈ each represent a methyl or ethyl radical or represent togetherand with the neighboring atom of oxygen, a heterocyclic radical such aspiperidino, morpholino, N'-methylpiperazino; R₃ represents the hydroxygroup, R₄ represents an atom of oxygen, R₅ represents the vinyl radical.3. The method defined in claim 1, wherein said labdane derivative isselected from the group consisting of:forskoline,7-O-deacetyl-forskoline, coleiorsine,7-O-deacetyl-7-β-O-propanoyl-forskoline,7-O-deacetyl-7-β-O-(ethoxycarbonyl)-forskoline,7-O-deacetyl-7-β-O-(propoxycarbonyl)-forskoline,7-O-deacetyl-7-β-O-(2,3-dihydroxy-propanoyl)-forskoline,7-O-deacetyl-7-β-O-(3-methyl-2-butenoyl)-forskoline,7,O-deacetyl-7-β-O-[4-(N'-methyl-piperazino)butanoyl-forskoline,7-O-deacetyl-7-β-O-(4-morpholino-butanoyl)-forskoline,6-β-O-[3-piperidino-propanoyl]-forskoline,6-β-O-(piperidino-acetyl)-forskoline.
 4. The method defined in claim 1,wherein the concentration of said derivative of labdane incorporated atleast in part in hydrated lipidic lamellar phases or liposomes, isincluded at a level of between 0.0001% and 1% by weight, with respect tothe total weight of the composition.
 5. The method defined in claim 1wherein said hydrated lipidic lamellar phases comprise liposomes.
 6. Themethod defined in claim 4 wherein said derivative of labdaneencapsulated at least in part in hydrated lipidic lamellar phases orliposome is included at a level of between about 0.01% and 0.1% byweight, with respect to the total weight of the composition.
 7. Themethod defined in claim 6 wherein said tyrosine ester is glucosetyrosinate. PG,37
 8. The process defined in claim 6 wherein saidxanthine is theophylline.
 9. The method defined in claim 6 wherein saidcomposition further comprises theophylline.
 10. The method defined inclaim 1, wherein said composition contains a pigment promoting amount ofat least one other active substance, selected from the group consistingof xanthines, tyrosine, tyrosine salts and tyrosine esters, said activesubstance being optionally incorporated at least in part in saidhydrated lipidic lamellar phases or liposomes.